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1.
Eur J Immunol ; 52(4): 566-581, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35092032

RESUMO

T-bet is the lineage-specifying transcription factor for CD4+ TH 1 cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.


Assuntos
Proteínas com Domínio T , Células Th1 , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Ativação Linfocitária , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismo , Células Th17/metabolismo , Células Th2
2.
J Immunol ; 206(11): 2725-2739, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34021046

RESUMO

Innate lymphoid cells are central to the regulation of immunity at mucosal barrier sites, with group 2 innate lymphoid cells (ILC2s) being particularly important in type 2 immunity. In this study, we demonstrate that microRNA(miR)-142 plays a critical, cell-intrinsic role in the homeostasis and function of ILC2s. Mice deficient for miR-142 expression demonstrate an ILC2 progenitor-biased development in the bone marrow, and along with peripheral ILC2s at mucosal sites, these cells display a greatly altered phenotype based on surface marker expression. ILC2 proliferative and effector functions are severely dysfunctional following Nippostrongylus brasiliensis infection, revealing a critical role for miR-142 isoforms in ILC2-mediated immune responses. Mechanistically, Socs1 and Gfi1 expression are regulated by miR-142 isoforms in ILC2s, impacting ILC2 phenotypes as well as the proliferative and effector capacity of these cells. The identification of these novel pathways opens potential new avenues to modulate ILC2-dependent immune functions.


Assuntos
Linfócitos/imunologia , MicroRNAs/imunologia , Animais , Células HEK293 , Homeostase , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética
3.
Colorectal Dis ; 25(7): 1489-1497, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37477408

RESUMO

This article adopts a multidisciplinary approach, including surgery, oncology, radiology and patient perspectives, to discuss the key points of debate surrounding a watch and wait approach. In an era of shared decision-making, discussion of watch and wait as an option in the context of complete clinical response is appropriate, although it is not the gold standard treatment. Key challenges are the difficulty in assessing for a complete clinical response, prediction of recurrence and access to timely diagnostics for surveillance. Salvage surgery has good results if regrowth is detected early but does have imperfect outcomes, with only a 90% salvage rate. Good communication with patients about the risks and alternatives is essential. Patients undergoing watch and wait should ideally be enrolled in prospective registries or clinical trials.


Assuntos
Terapia Neoadjuvante , Neoplasias Retais , Humanos , Estudos Prospectivos , Conduta Expectante , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias Retais/cirurgia , Quimiorradioterapia/métodos , Quimiorradioterapia Adjuvante , Equipe de Assistência ao Paciente , Resultado do Tratamento
4.
PLoS Genet ; 16(4): e1008583, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32236127

RESUMO

The precise control of eye size is essential for normal vision. TMEM98 is a highly conserved and widely expressed gene which appears to be involved in eye size regulation. Mutations in human TMEM98 are found in patients with nanophthalmos (very small eyes) and variants near the gene are associated in population studies with myopia and increased eye size. As complete loss of function mutations in mouse Tmem98 result in perinatal lethality, we produced mice deficient for Tmem98 in the retinal pigment epithelium (RPE), where Tmem98 is highly expressed. These mice have greatly enlarged eyes that are very fragile with very thin retinas, compressed choroid and thin sclera. To gain insight into the mechanism of action we used a proximity labelling approach to discover interacting proteins and identified MYRF as an interacting partner. Mutations of MYRF are also associated with nanophthalmos. The protein is an endoplasmic reticulum-tethered transcription factor which undergoes autoproteolytic cleavage to liberate the N-terminal part which then translocates to the nucleus where it acts as a transcription factor. We find that TMEM98 inhibits the self-cleavage of MYRF, in a novel regulatory mechanism. In RPE lacking TMEM98, MYRF is ectopically activated and abnormally localised to the nuclei. Our findings highlight the importance of the interplay between TMEM98 and MYRF in determining the size of the eye.


Assuntos
Olho/anatomia & histologia , Olho/metabolismo , Proteínas de Membrana/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Eletrorretinografia , Anormalidades do Olho/genética , Feminino , Deleção de Genes , Mutação com Perda de Função , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Tamanho do Órgão/genética , Ligação Proteica , Transporte Proteico , Epitélio Pigmentado da Retina/anormalidades , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
5.
Nat Immunol ; 11(9): 862-71, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20694009

RESUMO

In this study we demonstrate a new form of immunoregulation: engagement on CD4(+) T cells of the complement regulator CD46 promoted the effector potential of T helper type 1 cells (T(H)1 cells), but as interleukin 2 (IL-2) accumulated, it switched cells toward a regulatory phenotype, attenuating IL-2 production via the transcriptional regulator ICER/CREM and upregulating IL-10 after interaction of the CD46 tail with the serine-threonine kinase SPAK. Activated CD4(+) T cells produced CD46 ligands, and blocking CD46 inhibited IL-10 production. Furthermore, CD4(+) T cells in rheumatoid arthritis failed to switch, consequently producing excessive interferon-gamma (IFN-gamma). Finally, gammadelta T cells, which rarely produce IL-10, expressed an alternative CD46 isoform and were unable to switch. Nonetheless, coengagement of T cell antigen receptor (TCR) gammadelta and CD46 suppressed effector cytokine production, establishing that CD46 uses distinct mechanisms to regulate different T cell subsets during an immune response.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Regulação da Expressão Gênica , Proteína Cofatora de Membrana/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células CHO , Células Cultivadas , Enzimas Ativadoras do Complemento/imunologia , Cricetinae , Cricetulus , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/imunologia , Interleucina-2/imunologia , Células Jurkat , Linfócitos T Auxiliares-Indutores/imunologia
6.
Hum Mutat ; 42(10): 1239-1253, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34246199

RESUMO

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.


Assuntos
Albinismo Oculocutâneo , Estudo de Associação Genômica Ampla , Albinismo Oculocutâneo/diagnóstico , Albinismo Oculocutâneo/genética , Alelos , Humanos , Proteínas de Membrana Transportadoras/genética , Mutação
7.
Genet Med ; 23(3): 479-487, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33100333

RESUMO

PURPOSE: Albinism is a clinically and genetically heterogeneous condition. Despite analysis of the 20 known genes, ~30% patients remain unsolved. We aimed to identify new genes involved in albinism. METHODS: We sequenced a panel of genes with known or predicted involvement in melanogenesis in 230 unsolved albinism patients. RESULTS: We identified variants in the Dopachrome tautomerase (DCT) gene in two patients. One was compound heterozygous for a 14-bp deletion in exon 9 and c.118T>A p.(Cys40Ser). The second was homozygous for c.183C>G p.(Cys61Trp). Both patients had mild hair and skin hypopigmentation, and classical ocular features. CRISPR-Cas9 was used in C57BL/6J mice to create mutations identical to the missense variants carried by the patients, along with one loss-of-function indel. When bred to homozygosity the three mutations revealed hypopigmentation of the coat, milder for Cys40Ser compared with Cys61Trp or the frameshift mutation. Histological analysis identified significant hypopigmentation of the retinal pigmented epithelium (RPE) indicating that defective RPE melanogenesis could be associated with eye and vision defects. DCT loss of function in zebrafish embryos elicited hypopigmentation both in melanophores and RPE cells. CONCLUSION: DCT is the gene for a new type of oculocutaneous albinism that we propose to name OCA8.


Assuntos
Albinismo Oculocutâneo , Peixe-Zebra , Albinismo Oculocutâneo/genética , Animais , Humanos , Oxirredutases Intramoleculares , Camundongos , Camundongos Endogâmicos C57BL , Mutação
8.
Immunity ; 37(4): 674-84, 2012 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-23063332

RESUMO

Mice lacking the transcription factor T-bet in the innate immune system develop microbiota-dependent colitis. Here, we show that interleukin-17A (IL-17A)-producing IL-7Rα(+) innate lymphoid cells (ILCs) were potent promoters of disease in Tbx21(-/-)Rag2(-/-) ulcerative colitis (TRUC) mice. TNF-α produced by CD103(-)CD11b(+) dendritic cells synergized with IL-23 to drive IL-17A production by ILCs, demonstrating a previously unrecognized layer of cellular crosstalk between dendritic cells and ILCs. We have identified Helicobacter typhlonius as a key disease trigger driving excess TNF-α production and promoting colitis in TRUC mice. Crucially, T-bet also suppressed the expression of IL-7R, a key molecule involved in controlling intestinal ILC homeostasis. The importance of IL-7R signaling in TRUC disease was highlighted by the dramatic reduction in intestinal ILCs and attenuated colitis following IL-7R blockade. Taken together, these data demonstrate the mechanism by which T-bet regulates the complex interplay between mucosal dendritic cells, ILCs, and the intestinal microbiota.


Assuntos
Colite Ulcerativa/imunologia , Proteínas de Ligação a DNA/imunologia , Imunidade Inata , Linfócitos/imunologia , Receptores de Interleucina-7/imunologia , Proteínas com Domínio T/imunologia , Animais , Células Cultivadas , Doença Crônica , Colite Ulcerativa/microbiologia , Colite Ulcerativa/patologia , Proteínas de Ligação a DNA/deficiência , Helicobacter/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Transdução de Sinais , Proteínas com Domínio T/deficiência
10.
Am J Transplant ; 20(10): 2715-2727, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32277570

RESUMO

Organ transplantation is often lifesaving, but the long-term deleterious effects of combinatorial immunosuppression regimens and allograft failure cause significant morbidity and mortality. Long-term graft survival in the absence of continuing immunosuppression, defined as operational tolerance, has never been described in the context of multiple major histocompatibility complex (MHC) mismatches. Here, we show that miR-142 deficiency leads to indefinite allograft survival in a fully MHC mismatched murine cardiac transplant model in the absence of exogenous immunosuppression. We demonstrate that the cause of indefinite allograft survival in the absence of miR-142 maps specifically to the T cell compartment. Of therapeutic relevance, temporal deletion of miR-142 in adult mice prior to transplantation of a fully MHC mismatched skin allograft resulted in prolonged allograft survival. Mechanistically, miR-142 directly targets Tgfbr1 for repression in regulatory T cells (TREG ). This leads to increased TREG sensitivity to transforming growth factor - beta and promotes transplant tolerance via an augmented peripheral TREG response in the absence of miR-142. These data identify manipulation of miR-142 as a promising approach for the induction of tolerance in human transplantation.


Assuntos
Rejeição de Enxerto , MicroRNAs , Aloenxertos , Animais , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Linfócitos T Reguladores , Tolerância ao Transplante , Transplante Homólogo
11.
Am J Hum Genet ; 100(5): 706-724, 2017 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-28413018

RESUMO

During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.


Assuntos
Epilepsia/genética , Proteínas/genética , Espasmos Infantis/genética , Transmissão Sináptica , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Modelos Animais de Doenças , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipagem , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Conformação Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantis/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismo
12.
Development ; 142(4): 620-32, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25670789

RESUMO

Melanocyte development provides an excellent model for studying more complex developmental processes. Melanocytes have an apparently simple aetiology, differentiating from the neural crest and migrating through the developing embryo to specific locations within the skin and hair follicles, and to other sites in the body. The study of pigmentation mutations in the mouse provided the initial key to identifying the genes and proteins involved in melanocyte development. In addition, work on chicken has provided important embryological and molecular insights, whereas studies in zebrafish have allowed live imaging as well as genetic and transgenic approaches. This cross-species approach is powerful and, as we review here, has resulted in a detailed understanding of melanocyte development and differentiation, melanocyte stem cells and the role of the melanocyte lineage in diseases such as melanoma.


Assuntos
Melanócitos/citologia , Melanócitos/metabolismo , Animais , Humanos , Melanoma/metabolismo , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Crista Neural/citologia , Células-Tronco/citologia , Células-Tronco/metabolismo
13.
Nature ; 549(7672): 337-339, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28869972
14.
Nature ; 477(7364): 289-94, 2011 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-21921910

RESUMO

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism.


Assuntos
Regulação da Expressão Gênica/genética , Variação Genética/genética , Genoma/genética , Camundongos Endogâmicos/genética , Camundongos/genética , Fenótipo , Alelos , Animais , Animais de Laboratório/genética , Genômica , Camundongos/classificação , Camundongos Endogâmicos C57BL/genética , Filogenia , Locos de Características Quantitativas/genética
15.
PLoS Genet ; 10(10): e1004688, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25356849

RESUMO

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.


Assuntos
Proteínas de Transporte de Ânions/genética , Cóclea/patologia , Orelha Interna/patologia , Perda Auditiva/genética , Idade de Início , Animais , Proteínas de Transporte de Ânions/deficiência , Proteínas de Transporte de Ânions/metabolismo , Segmento Anterior do Olho/metabolismo , Segmento Anterior do Olho/patologia , Cóclea/metabolismo , Conexina 26 , Conexinas , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva/metabolismo , Perda Auditiva/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Organogênese/genética , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Estria Vascular/patologia , Peixe-Zebra
16.
PLoS Genet ; 10(5): e1004359, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24809698

RESUMO

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.


Assuntos
Genes Dominantes , Genes Letais , Glaucoma/genética , Proteínas com Homeodomínio LIM/genética , Fatores de Transcrição/genética , Alelos , Animais , Padronização Corporal , Dimerização , Heterozigoto , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto
17.
PLoS Genet ; 10(9): e1004577, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25232951

RESUMO

Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.


Assuntos
Cílios/metabolismo , Cílios/fisiologia , Proteínas/metabolismo , Animais , Dineínas do Axonema , Axonema/genética , Axonema/metabolismo , Sítios de Ligação/genética , Linhagem Celular , Pré-Escolar , Cílios/genética , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/metabolismo , Drosophila/genética , Drosophila/metabolismo , Dineínas/genética , Dineínas/metabolismo , Feminino , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Masculino , Mutação/genética , Linhagem , Fenótipo , Proteínas/genética , Transcrição Gênica/genética
18.
PLoS Genet ; 9(12): e1003928, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24415959

RESUMO

Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1(Gt/Gt)). Surprisingly, Azi1(Gt/Gt) MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease.


Assuntos
Cílios/genética , Infertilidade Masculina/genética , Proteínas/genética , Cauda do Espermatozoide/patologia , Animais , Proteínas de Ciclo Celular , Centríolos/genética , Centríolos/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Flagelos/metabolismo , Flagelos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Infertilidade Masculina/etiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteínas/metabolismo , RNA Interferente Pequeno
19.
PLoS Genet ; 9(4): e1003453, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23637623

RESUMO

Following domestication, livestock breeds have experienced intense selection pressures for the development of desirable traits. This has resulted in a large diversity of breeds that display variation in many phenotypic traits, such as coat colour, muscle composition, early maturity, growth rate, body size, reproduction, and behaviour. To better understand the relationship between genomic composition and phenotypic diversity arising from breed development, the genomes of 13 traditional and commercial European pig breeds were scanned for signatures of diversifying selection using the Porcine60K SNP chip, applying a between-population (differentiation) approach. Signatures of diversifying selection between breeds were found in genomic regions associated with traits related to breed standard criteria, such as coat colour and ear morphology. Amino acid differences in the EDNRB gene appear to be associated with one of these signatures, and variation in the KITLG gene may be associated with another. Other selection signals were found in genomic regions including QTLs and genes associated with production traits such as reproduction, growth, and fat deposition. Some selection signatures were associated with regions showing evidence of introgression from Asian breeds. When the European breeds were compared with wild boar, genomic regions with high levels of differentiation harboured genes related to bone formation, growth, and fat deposition.


Assuntos
Cruzamento , Sus scrofa , Animais , Genoma , Genômica , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Seleção Genética , Sus scrofa/genética , Suínos
20.
PLoS Genet ; 9(12): e1003998, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24348270

RESUMO

Mp is an irradiation-induced mouse mutation associated with microphthalmia, micropinna and hind limb syndactyly. We show that Mp is caused by a 660 kb balanced inversion on chromosome 18 producing reciprocal 3-prime gene fusion events involving Fbn2 and Isoc1. The Isoc1-Fbn2 fusion gene (Isoc1(Mp)) mRNA has a frameshift and early stop codon resulting in nonsense mediated decay. Homozygous deletions of Isoc1 do not support a significant developmental role for this gene. The Fbn2-Isoc1 fusion gene (Fbn2 (Mp)) predicted protein consists of the N-terminal Fibrillin-2 (amino acids 1-2646, exons 1-62) lacking the C-terminal furin-cleavage site with a short out-of-frame extension encoded by the final exon of Isoc1. The Mp limb phenotype is consistent with that reported in Fbn2 null embryos. However, severe eye malformations, a defining feature of Mp, are not seen in Fbn2 null animals. Fibrillin-2(Mp) forms large fibrillar structures within the rough endoplasmic reticulum (rER) associated with an unfolded protein response and quantitative mass spectrometry shows a generalised defect in protein secretion in conditioned media from mutant cells. In the embryonic eye Fbn2 is expressed within the peripheral ciliary margin (CM). Mp embryos show reduced canonical Wnt-signalling in the CM - known to be essential for ciliary body development - and show subsequent aplasia of CM-derived structures. We propose that the Mp "worse-than-null" eye phenotype plausibly results from a failure in normal trafficking of proteins that are co-expressed with Fbn2 within the CM. The prediction of similar trans-acting protein effects will be an important challenge in the medical interpretation of human mutations from whole exome sequencing.


Assuntos
Anormalidades do Olho/genética , Proteínas dos Microfilamentos/genética , Microftalmia/genética , Mutação/efeitos da radiação , Animais , Inversão Cromossômica/genética , Cromossomos Humanos Par 18/genética , Éxons , Olho/crescimento & desenvolvimento , Olho/fisiopatologia , Anormalidades do Olho/fisiopatologia , Fibrilina-2 , Fibrilinas , Mutação da Fase de Leitura , Humanos , Camundongos , Microftalmia/fisiopatologia , Fenótipo , Sindactilia/genética , Sindactilia/fisiopatologia , Via de Sinalização Wnt/genética
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